The subject of the present invention is a new device for the culturing of plant or animal tissue or organism cultures (in the following called tissues), as well as culturing processes with the use of such devices.
The culturing of cells for the production of cell material and especially of metabolic products of these cells is of continuous importance since the chemical synthesis of such products is frequently difficult or impossible or proves to be uneconomical in comparison with the biochemical production. Besides the already long known culturing to a wide extent of yeasts, moulds and bacteria, the culturing of plant and animal cells is also to an increasing extent of importance for the production of certain products. The investigation of suitable culturing conditions and possibly the genetic change and culturing of plant and animal cells occurring in nature for the achievement of high yields are thereby urgent objectives of research.
One differentiates plant or animal cell and tissue cultures (preferably cell suspensions, callus cultures) from plant organ cultures (transformed root hair (hairy root), root or shoot cultures (shooty teratomas)). For the fermentation, hitherto there were used, above all, cell suspension cultures which consist of undifferentiated individual cells or cell aggregates. Since the 1980""s, intensive efforts have been underway to culture these cell suspension cultures in large scale fermenters and to use them for the commercial production of tissue component materials.
However, apart from a few exceptions, it has been shown that cell suspension cultures are genetically frequently unstable (somaclonal variation). This has a serious influence on the active material production since even promising high capacity cell lines can, after a few cycles, be subject to great variations or the production of the active materials is completely suppressed. One has attributed this instability to the influence of growth regulators, as well as to the undifferentiated state of the cells which are frequently revealed as not stable without sufficient differentiation of cell organelles or without corresponding cell-cell contact of individual biosynthesis routes. Thus, epigenetic factors play a quite important influence for the biosynthesis.
Plant organ cultures and here, above all, the commercially interesting transfonned root hair cultures form differentiated tissues which also in long-term cultures prove to be genetically clearly more stable than cell suspension cultures. Thanks to the rapid growth of transformed root hair cultures, which frequently display comparable growth rates to cell suspension cultures, these organ cultures are suitable for the fermentative production of commercially interesting component materials. Above all, transformed root hair culturesxe2x80x94other than cell suspension culturesxe2x80x94can be cultured without growth regulators. By way of example, reference is made to the synthesis of taxoids, podophyllotoxins or rosmaric acids which are produced by such processes. Individual growth regulators can inhibit the biosynthesis of secondary metabolites.
However, plant and animal organ cultures require a completely new fermenter design since a scale-up of the laboratory installations makes difficult the gas and nutrient solution provision due to the inhomogeneous tissues which cannot be mixed up.
For the culturing of tissues, it is necessary regularly to supply to the cells with mineral materials, growth regulators, carbon sources, normally saccharose, fructose or glucose, as well as possibly gases, such as oxygen or carbon dioxide, necessary for the nutrition of the cells.
The simplest and most economic form of the culturing of cells is the suspension culture, whereby isolated cells are suspended in a nutrient liquid, consumed nutrient components are regularly supplied to the nutrient solution and possibly a gassing is carried out for the maintenance of the suspension and nutrition of the cells. By means of appropriate growth regulators, a growing together of the cells to give comparatively large aggregates is prevented. It thereby proves to be disadvantageous that many plant or animal cells are not viable for a long time in this form and the formation of metabolites which are difficult to eliminate from the culture liquid requires a frequent transfer of the cells into fresh nutrient solution.
For smaller cultures, it is possible to inoculate the cells on to the surface of nutrient-containing gels, for example on to Petri dishes containing agar, whereby the cells take up the necessary nutrient components from the gel and the surrounding atmosphere. In the case of this method, too, it proves to be disadvantageous that under these conditions, many cells grow poorly and only slowly and metabolites separate out which, in part, enrich in the surrounding medium and again slow down the growth of the cells and the cell division or even kill off the cells. By means of the fixing on the gel surfaces, the cells grow to comparatively large heaps which, in turn, has the disadvantage that only the surface is in contact with the gas and the lower side with the nutrient liquid and thus slows down the nutrient supply in the case of growth. Here, too, in order to maintain a sufficient growth, a frequent inoculating over of the cultures to new nutrient bases is necessary.
In order to avoid the suspension culture of isolated cells, one has, therefore, changed over to culturing differentiated cultures, such as xe2x80x9chairy rootsxe2x80x9d or plant shoots or leaf tissue. In suspension, such comparatively large aggregates tend to dehomogenisation, especially in comparatively large reactors, whereby a change or adaptation of the process conditions was necessary by means of which a uniform supplying of the cell aggregates with nutrient solution and the necessary gases is achieved.
A widely used process is based on the fixing of the tissues to solid carriers and allowing a thin layer of nutrient liquid to run over the carrier so that the tissues are continuously supplied with fresh nutrient solution and, at the same time, possess a sufficient contact to the gas atmosphere in the reactor. As carrier bodies there are used plates or fabrics, especially mesh grids or rod constructions, as substrate which are arranged at a distance parallel to one another in appropriate reactors so that the intermediate spaces make possible an impingement with nutrient liquid and gas and a certain growth of the cell culture. The fixing of the cells on the carrier thereby takes place e.g. by squeezing into the gaps or angles of the carrier surface (cf. EP 234 868 and U.S. Pat No. 5,585,266). For the fermentation of so-called xe2x80x9chairy root culturesxe2x80x9d, the use of fermenters which contain a system of taut wires is especially recommended, whereby the distance of the wires at the crossing points is so small that the plant tissue is firmly held at these points or, by special formation of the wires with spikes, are firmly held in these spike axes (cf. WO 85/10958).
The fermentation vessels of the prior art usually consist, for reasons of stability, of metal, for example steel or aluminum sheet, or, because of transparency, of glass or acrylic glass and preponderantly have a cubic shape which makes possible a space-saving arrangement of several parallel carrier plate systems in the interior. A removable lid to which, possibly besides the inlet pipes, are also fixed the carrier plates, permits an access to the container, especially for harvesting of the cell culture and for cleaning. The production of the fermentation vessels from stable materials permits such reactors to be arranged fixedly next to one another on the bottom but has the disadvantage that the production and servicing of such vessels is expensive and does not permit an adaptation to the size of the culture.
Therefore, the task exists to find simple and economic devices for the culturing of tissues, especially of plant tissues, which are simple and economic to produce, are alterable in size and permit a simple handling and servicing.
In particular, it is an objective of the present invention, with the use of such fermentation devices, to make available a process for the culturing of plant tissue of the xe2x80x9chairy root typexe2x80x9d or xe2x80x9cplant shoot typexe2x80x9d.
The present invention provides a device for the culturing of plant and animal tissue, comprising a fermenter vessel, an inlet for liquid nutrients and gases, as well as devices for removal of spent nutrient liquids and gases and solid carrier plates for the tissue which are permeable for the said liquid nutrients and gases and are fixedly arranged within the fermenter vessel, whereby the fermenter vessel comprises a form-stable lid part which carries the various inlet and outlet pipes as well as the carrier plates for the tissue and comprises a cup- or bag-shaped plastic tube, the opening of which is sealed off and fixed against the lid.
According to one embodiment 3-8 carrier plates are radially fixed on a central holding means and the supply of the nutrient solution takes place via 3-8 supply pipes present in the outer region between the plates.
According to a further embodiment the supply pipes contain bores as nozzles for the outlet of the nutrient solution and gas inlet pipes are guided parallel to the pipes which, in appropriate places, also contain bores from which gas emerges.
It is preferred that in the outer region between the carrier plates are arranged one or more sucking-off pipes for spent nutrient liquid and spent gas which on the end contain a sucking-off nozzle.
In another embodiment the inlet pipe represents a central pipe which comprises two or more tube pieces combined via intermediate adapters, whereby the adapter contains nozzles for the atomization of the nutrient solution and the tube pieces carry 3-8 radially projecting lattice plates as carrier plates for the cells, whereby the length of the assembled supply pipe corresponds to the length of the plastic tube of the fermenter vessel and the cross-section through the carrier lattice corresponds to the opening of the plastic tube or to the size of the lid.
It is preferred that, in the outer region between the carrier plates, one or more sucking-off pipes for spent nutrient liquid and spent gases are arranged which also comprises tube pieces which correspond in length to the tube pieces of the supply pipe and contain adapter pieces for the connecting of the tube pieces, as well as a sucking-off nozzle on the end, whereby the length of this pipe corresponds to the length of the plastic tube.
The carrier plates are arranged transversely to the longitudinal axis of the reactor or/and can be adjusted on a central holding means via tilt devices at an angle of 0-90xc2x0 to the holding means.
In a further embodiment one or more devices are connected via appropriate pipe connections and holding devices to a central supply system. It is preferred that the nutrient solution is circulated via a nutrient solution tank in the form of a plastic sack.